Avian Eggshell Research Articles

Nature's Palette: Characterization of Shared Pigments in Colorful Avian and Mollusk Shells.

Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature’s palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa.

Secretary’s Report, 2015

Secretary’s Report, 2015

The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata).

BackgroundThe proteins of avian eggshell organic matrices are thought to control the mineralization of the eggshell in the shell gland (uterus). Proteomic analysis of such matrices identified many candidates for such a role. However, all matrices analyzed to date come from species of one avian family, the Phasianidae. To analyze the conservation of such proteins throughout the entire class Aves and to possibly identify a common protein toolkit enabling eggshell mineralization, it is important to analyze eggshell matrices from other avian families. Because mass spectrometry-based in-depth proteomic analysis still depends on sequence databases as comprehensive and accurate as possible, the obvious choice for a first such comparative study was the eggshell matrix of zebra finch, the genome sequence of which is the only songbird genome published to date.ResultsThe zebra finch eggshell matrix comprised 475 accepted protein identifications. Most of these proteins (84 %) were previously identified in species of the Phasianidae family (chicken, turkey, quail). This also included most of the so-called eggshell-specific proteins, the ovocleidins and ovocalyxins. Ovocleidin-116 was the second most abundant protein in the zebra finch eggshell matrix. Major proteins also included ovocalyxin-32 and -36. The sequence of ovocleidin-17 was not contained in the sequence database, but a presumptive homolog was tentatively identified by N-terminal sequence analysis of a prominent 17 kDa band. The major proteins also included three proteins similar to ovalbumin, the most abundant of which was identified as ovalbumin with the aid of two characteristic phosphorylation sites. Several other proteins identified in Phasianidae eggshell matrices were not identified. When the zebra finch sequence database contained a sequence similar to a missing phasianid protein it may be assumed that the protein is missing from the matrix. This applied to ovocalyxin-21/gastrokine-1, a major protein of the chicken eggshell matrix, to EDIL3 and to lactadherin. In other cases failure to identify a particular protein may be due to the absence of this protein from the sequence database, highlighting the importance of better, more comprehensive sequence databases.ConclusionsThe results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins. All the more important it is to elucidate the role of these proteins at the molecular level. This cannot be achieved by proteomic studies but will need application of other methods, such as atomic force microscopy or gene knockouts. However, it will also be important to analyze more eggshell matrices of different avian families to unequivocally identify other mineralization toolkit proteins apart from ovocleidins and ovocalyxins. Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases. The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0086-1) contains supplementary material, which is available to authorized users.

Hierarchical structure and mechanical properties of snake (Naja atra) and turtle (Ocadia sinensis) eggshells

After hundreds of million years of evolution, natural armors have evolved in various organisms, and has manifested in diverse forms such as eggshells, abalone shells, alligator osteoderms, turtle shells, and fish scales. Eggshells serve as multifunctional shields for successful embryogenesis, such as protection, moisture control and thermal regulation. Unlike calcareous avian eggshells which are brittle and hard, reptilians have leathery eggshells that are tough and flexible. Reptilian eggshells can withstand collision damages when laid in holes and dropped onto each other, and reduce abrasion caused by buried sand. In this study, we investigate structure and mechanical properties of eggshells of Taiwan cobra snake (Naja atra) and Chinese striped-neck turtle (Ocadia sinensis). From Acid Fuchsin Orange G (AFOG) staining and ATR-FTIR examination, we found that both eggshells are mainly composed of keratin. The mechanical properties of demineralized snake and turtle eggshells were evaluated by tensile and fracture tests and show distinctly difference. Turtle eggshells are relatively stiff and rigid, while snake eggshells behave as elastomers, which are highly extensible and reversible. The exceptional deformability (110–230% tensile strain) and toughness of snake eggshells are contributed by the wavy and random arrangement of keratin fibers as well as collagen layers. Multi-scale toughening mechanisms of snake eggshells were observed and elucidated, including crack deflection and twisting, fibers reorientation, sliding and bridging, inter-laminar shear effect, as well as the α–β phase transition of keratin. Inspirations from the structural and mechanical designs of reptilian eggshells may lead to the synthesis of tough, extensible, lightweight composites which could be further applied in the flexible devices, packaging and bio-medical fields. Statement of significanceAmniotic eggshells serve as multifunctional shields for successful embryogenesis. The avian eggshells have been extensively studied while there are very few studies on reptilian eggshells and most of them focused on mineralization and embryotic development. For the first time, the hierarchical structure and mechanical properties of snake and turtle eggshells are comprehensively and comparatively studied. Both snake and turtle eggshells are multilayer, hierarchically-structured composites consisting mainly of keratin yet their mechanical behaviors are distinctly different. Turtle eggshells are stiff and rigid, while snake eggshells are highly extensible (>200%) and reversible due to multiple deformation stages, phase transition of keratin and various toughening mechanisms. We believe that this study will make positive scientific impact and interest the broad and multidisciplinary readership.

Comparison of the structure, crystallography and composition of eggshells of the guinea fowl and graylag goose

The structure and composition of the eggshells of two commercial species (guinea fowl and greylag goose) have been studied. Thin sections and scanning electron microcopy show the similarity of the overall structure, but the relative thickness of the layers differs in these two taxa. Atomic force microscopy shows that the different layers are composed of rounded, heterogeneous granules, the diameter of which is between 50 and 100nm, with a thin cortex. Infrared data and thermogravimetric analyses show that both eggshells are made of calcite, but differing on the quality and quantity when the organic component is considered. Chemical maps show that chemical element distribution is not uniform within a sample, and differs between the species, but with low magnesium content. Electron back scattered diffraction confirms the eggshells are calcite, but the microtexture strongly differs between the two species. Based on the chemical-structural differences, a species-specific biological control on the biomineralization is found, despite the rapid formation of an eggshell. Overall results indicate that to estimate the quality of eggshells, such as resistance to breakage, is not a straightforward process because of the high complexity of avian eggshell biomineralization.

Identifying specific proteins involved in eggshell membrane formation using gene expression analysis and bioinformatics

BackgroundThe avian eggshell membranes surround the egg white and provide a structural foundation for calcification of the eggshell which is essential for avian reproduction; moreover, it is also a natural biomaterial with many potential industrial and biomedical applications. Due to the insoluble and stable nature of the eggshell membrane fibres, their formation and protein constituents remain poorly characterized. The purpose of this study was to identify genes encoding eggshell membrane proteins, particularly those responsible for its structural features, by analyzing the transcriptome of the white isthmus segment of the oviduct, which is the specialized region responsible for the fabrication of the membrane fibres.ResultsThe Del-Mar 14 K chicken microarray was used to investigate up-regulated expression of transcripts in the white isthmus (WI) compared with the adjacent magnum (Ma) and uterine (Ut) segments of the hen oviduct. Analysis revealed 135 clones hybridizing to over-expressed transcripts (WI/Ma + WI/Ut), and corresponding to 107 NCBI annotated non-redundant Gallus gallus gene IDs. This combined analysis revealed that the structural proteins highly over-expressed in the white isthmus include collagen X (COL10A1), fibrillin-1 (FBN1) and cysteine rich eggshell membrane protein (CREMP). These results validate previous proteomics studies which have identified collagen X (α-1) and CREMP in soluble eggshell extracts. Genes encoding collagen-processing enzymes such as lysyl oxidase homologs 1, 2 and 3 (LOXL1, LOXL2 and LOXL3), prolyl 4 hydroxylase subunit α-2 and beta polypeptide (P4HA2 and P4HB) as well as peptidyl-prolyl cis-trans isomerase C (PPIC) were also over-expressed. Additionally, genes encoding proteins known to regulate disulfide cross-linking, including sulfhydryl oxidase (QSOX1) and thioredoxin (TXN), were identified which suggests that coordinated up-regulation of genes in the white isthmus is associated with eggshell membrane fibre formation.ConclusionsThe present study has identified genes associated with the processing of collagen, other structural proteins, and disulfide-mediated cross-linking during eggshell membrane formation in the white isthmus. Identification of these genes will provide new insight into eggshell membrane structure and mechanisms of formation that will assist in the development of selection strategies to improve eggshell quality and food safety of the table egg.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2013-3) contains supplementary material, which is available to authorized users.

Quantitative proteomics analysis of eggshell membrane proteins during chick embryonic development

The avian eggshell membrane (ESM) is a meshwork made up of highly cross-linked protein fibers and it is a scaffold upon which biomineralization of the eggshell is initiated. The ESM and associated shell participates in embryonic development by providing physical and chemical protection against pathogen invasion. We performed quantitative proteomic analysis of ESM proteins on multiple days during the three phases of embryonic development. The ESMs were stripped from both fertilized and unfertilized eggs at different days of incubation, and solubilized in a novel manner using TCEP-HCl (Tris (2-carboxyethyl) phosphine hydrochloride). The changes in ESM proteins between occurred during incubation were analyzed. Bioinformatics analysis revealed that of the 12 functional protein clusters identified, protease inhibitors were present at all phases of chick development. A group of proteins involved in calcium binding and oxygen transport were only present during the second phase. Extracellular matrix, cell adhesion proteins related to the vascularization of chorioallantoic membrane (CAM), antimicrobial proteins and proteins involved in the binding and transport of lipids were found in the second and third phases of development. These findings provide insight into the functionality and evolving nature of ESM associated proteins involved in chick embryonic development. Biological significanceThe eggshell membranes (ESMs) are a fibrous scaffold that consists of highly crosslinked collagens (types I, V but mainly X), glycoproteins and CREMPs (cysteine-rich eggshell membrane proteins). The ESMs aid in the development of the chick embryo and protect it against pathogen invasion. This biopolymeric fibrous net functions as a platform for nucleation of the calcitic eggshell which provides a primary physical barrier against bacterial ingress. Comparative proteomic analyses of proteins in the ESMs from fertilized eggs and unfertilized eggs showed changes in their levels which varied between the specific phases of chick embryogenesis across 19days of incubation. Bioinformatics characterization of these ESM proteins provides understanding of their evolving nature during chick embryonic development.

Variation in Mesoscopic Textures of Biogenic and Biomimetic Calcite Crystals

Mesoscale granular textures having a single-crystalline feature are widely observed on various biogenic and biomimetic calcite crystals, although the distribution of organic phases and the magnitude of lattice strain in the textured crystals vary with organism species or growth conditions of the biomimetic process. The prismatic layer of a fan mussel exhibits a relatively homogeneous, low-strain texture consisting of nanoscale grains with discrete organic inclusions; the prism structures of a pearl oyster and an avian eggshell have a high-strain granular texture with localized organic phases. The high-strain granular textures were artificially produced through the mesoscopic dendritic growth of calcite by the physical impedance of a rigid gel matrix. Facet growth of the crystal involving nanoscale segregation of soluble polymers in a supersaturated solution would result in the formation of the low-strain body having mesoscopic textures.

The cuticle modulates ultraviolet reflectance of avian eggshells.

ABSTRACTAvian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour.

Not so colourful after all: eggshell pigments constrain avian eggshell colour space.

Birds' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08-0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models' predictions can be improved by accounting for the reflectance of the eggshell's calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity.

Amorphous calcium carbonate controls avian eggshell mineralization: A new paradigm for understanding rapid eggshell calcification.

Avian eggshell mineralization is the fastest biogenic calcification process known in nature. How this is achieved while producing a highly crystalline material composed of large calcite columnar single crystals remains largely unknown. Here we report that eggshell mineral originates from the accumulation of flat disk-shaped amorphous calcium carbonate (ACC) particles on specific organic sites on the eggshell membrane, which are rich in proteins and sulfated proteoglycans. These structures known as mammillary cores promote the nucleation and stabilization of a amorphous calcium carbonate with calcitic short range order which predetermine the calcite composition of the mature eggshell. The amorphous nature of the precursor phase was confirmed by the diffuse scattering of X-rays and electrons. The nascent calcitic short-range order of this transient mineral phase was revealed by infrared spectroscopy and HRTEM. The ACC mineral deposited around the mammillary core sites progressively transforms directly into calcite crystals without the occurrence of any intermediate phase. Ionic speciation data suggest that the uterine fluid is equilibrated with amorphous calcium carbonate, throughout the duration of eggshell mineralization process, supporting that this mineral phase is constantly forming at the shell mineralization front. On the other hand, the transient amorphous calcium carbonate mineral deposits, as well as the calcite crystals into which they are converted, form by the ordered aggregation of nanoparticles that support the rapid mineralization of the eggshell. The results of this study alter our current understanding of avian eggshell calcification and provide new insights into the genesis and formation of calcium carbonate biominerals in vertebrates.

Quantitative interior x-ray nanotomography by a hybrid imaging technique

Hierarchical structures appear often in life and materials sciences, and their characterization profits greatly from imaging methods that allow seamless probing of various length scales without sacrificing image quality. X-ray tomography is particularly adept at probing 3D structures; however, zooming in on a region of interest results in a loss of quantitativeness of image contrast and suffers from artifacts unless a priori knowledge or assumptions about the sample are used. Here, we demonstrate a hybrid technique that exploits a micrometer-resolution overview to realize ab initio nanoscale interior tomography with quantitative contrast. In a study of avian eggshell, a model for bionanoporous materials, our approach reveals a complex arrangement of vesicles with sizes ranging from hundred nanometers to a few micrometers. We anticipate that such an approach can be widely adopted and benefited from at synchrotron and laboratory sources, for instance, where such zooming capabilities are already present or can be readily realized.

A nanostructural basis for gloss of avian eggshells.

The role of pigments in generating the colour and maculation of birds' eggs is well characterized, whereas the effects of the eggshell's nanostructure on the visual appearance of eggs are little studied. Here, we examined the nanostructural basis of glossiness of tinamou eggs. Tinamou eggs are well known for their glossy appearance, but the underlying mechanism responsible for this optical effect is unclear. Using experimental manipulations in conjunction with angle-resolved spectrophotometry, scanning electron microscopy, atomic force microscopy and chemical analyses, we show that the glossy appearance of tinamou eggshells is produced by an extremely smooth cuticle, composed of calcium carbonate, calcium phosphate and, potentially, organic compounds such as proteins and pigments. Optical calculations corroborate surface smoothness as the main factor producing gloss. Furthermore, we reveal the presence of weak iridescence on eggs of the great tinamou (Tinamus major), an optical effect never previously documented for bird eggs. These data highlight the need for further exploration into the nanostructural mechanisms for the production of colour and other optical effects of avian eggshells.

Transparent soft PDMS eggshell

In vivo 3D fluorescent image remains a technological barrier for biologists and clinical scientists although green fluorescent protein (GFP) imaging has long been performed rather well at cellular level. Meanwhile, robust enough portable devices are also challenging lab-on-a-chip advocators who wish their designs to be nurtured by the end users. This work is dedicated to propose a conceptually innovated transparent soft PDMS avian eggshell to directly tackle the above two goals. Here, an “egg-on-a-chip” scheme is originally developed and demonstrated by a newly developed PDMS “soft” process method. Unlike its ancestor-the conventional “lab-on-a-chip” (LOC) which is basically chemically based, the current “egg-on-a-chip”, intrinsically inherited with biological natures, opens a way to integrate biological parts or whole system in a miniature sized device. Such biomimics system contains much condensed environmental evolutional tensor inside than those of the existing LOC compacted with artificial components which however are quite difficult to incorporate various life factors inside. Owning unique advantages, a series of transparent PDMS whole “eggshells” have been fabricated and applied to culture avian embryos up to 17.5 days and chimeric eggshells were engineered on normal eggs. In addition, X-stage embryos were successfully initiated in such system and pre-chorioallantoic membrane was observed. Further, limitation of the present process was interpreted and potential approach to improve it was suggested. With both high optical transparency and engineering subtlety fully integrated together, the present method not only provides an ideal transparent imaging platform for studying functional embryo development including life mystery, but also promises a future strategy for “lab-on-an-egg” technology which may be important in a wide variety of either fundamental or practical areas.

Novel identification of matrix proteins involved in calcitic biomineralization.

Biomineralization is essential for the development of hard tissues in vertebrates, which includes both calcium phosphate and calcium carbonate structures. Calcitic mineralization by calcium carbonate is an important process in the formation of otoconia, which are gravity receptor organs located in the inner ear and are responsible for balance and for sensing linear acceleration. Deficiencies in the regulation of their biomineralization can lead to otoconia degeneration and eventually benign paroxysmal position vertigo (BPPV), which is the main cause of vertigo in humans. Eggshell formation in chicken is one of the fastest known biomineralization processes and is an excellent model for the study of calcitic biomineralization. Cross-analysis of proteomic data from two mineralized models, fertilized and unfertilized chicken eggshells, identified proteins associated with the mammillary cones that are the sites of initiation of eggshell formation. We hypothesize that these proteins regulate the earliest stages of eggshell calcification. The human homologs of these proteins are therefore potential candidates to play a role in otoconia biomineralization.

Analysing avian eggshell pigments with Raman spectroscopy.

Avian eggshells are variable in appearance, including coloration. Here, we demonstrate that Raman spectroscopy can provide accurate diagnostic information about major eggshell constituents, including the pigments biliverdin and protoporphyrin IX. Eggshells pigmented with biliverdin showed a series of pigment-diagnostic Raman peaks under 785 nm excitation. Eggshells pigmented with protoporphyrin IX showed strong emission under 1064 nm and 785 nm excitation, whereas resonance Raman spectra (351 nm excitation) showed a set of protoporphyrin IX informative peaks characteristic of protoporphyrin IX. As representative examples, we identified biliverdin in the olive green eggshells of elegant crested tinamous (Eudromia elegans) and in the blue eggshells of extinct upland moa (Megalapteryx didinus). This study encourages the wider use of Raman spectroscopy in pigment and coloration research and highlights the value of this technique for non-destructive analyses of museum eggshell specimens.

Visualisation of Latent Fingerprint on Wild Bird Eggshells by Alternate Light Sources Following Superglue Fuming

The theft of the eggs of endangered or protected species of bird, and subsequent reduction in wildlife population, is a significant problem worldwide. Detection rates are comparatively low towards this type of crime and fingerprinting of egg shells is infrequently utilised due to the technical barrier. This paper explores a novel technique using cyanoacrylate (superglue) fuming in conjunction with fluorescent dye to visualise latent fingerprints upon avian eggshells assisted with alternate light sources. A systematic investigation of experimental parameters has also been carried out to optimise the condition for the fingerprint visualisation. This research project has successfully developed latent fingerprints upon smooth wild bird eggshells but was less successful on developing prints on more textured, porous eggshells.

Corrosion casts: A novel application of a polyurethane resin (PU4ii) for visualizing eggshell pore morphology

Avian eggshells serve the dual purposes of protecting the developing embryo from the external environment while also facilitating the loss of water vapor and the required exchange of CO2 and O2 gases. Pores that span the eggshell enable the loss of water and trans-shell gas exchange. Although knowledge of the geometry of these spaces is necessary to generate accurate estimates of the rate of gas diffusion across the shell, few techniques exist to obtain these data. Estimates of gas conductance across eggshells are typically calculated from eggshell thickness and the size and number of the pores on the exterior eggshell surface; the trans-shell pore spaces are assumed to be cylindrical in shape. To enable the testing of this assumption, we devised a novel method to visualize the three-dimensional morphology of eggshell pores using PU4ii, a polyurethane-based resin. Casts of the pores of eggshells of the domestic chicken (Gallus gallus )a nd House Sparrow (Passer domesticus) were unbranched and varied in diameter throughout their length, while casts of the pores of eggshells of the Ostrich (Struthio camelus) revealed a complex network of interconnected spaces. The simplicity of this technique and the stability and resilience of the resulting casts provide opportunities to predict gas flux across the shell and to evaluate the morphology of eggshell pores among birds from different taxonomic groups.

Proteomic analysis of chicken eggshell cuticle membrane layer.

The eggshell is a barrier that plays an important role in the defense of the egg against microbial and other infections; it protects the developing bird against unfavorable impacts of the environment and is essential for the reproduction of birds. The avian eggshell is a complex structure that is formed during movement along the oviduct by producing a multilayered mineral-organic composite. The extractable proteins of avian eggshells have been studied extensively and many of them identified, however, the insoluble (non-extractable) proteins have been sparsely studied. We studied the EDTA-insoluble proteinaceous film from the cuticle layer of eggshell. This film consists of three main areas: spots (cca 300 μm diameter), blotches (small spots with diameter only tens of μm), and the surroundings (i.e., the area without spots and blotches) where spots contain a visible accumulation of pigment. These areas were cut out of the membrane by laser microdissection, proteins were cleavaged by trypsin, and the peptides were analyzed by nLC/MS (Q-TOF). This study has identified 29 proteins and a further eight were determined by less specific "cleavage" with semitrypsin. The relative abundances of these proteins were determined using the exponentially modified protein abundance index (emPAI) where the most dominant proteins were eggshell-specific ones, such as ovocleidin-17 and ovocleidin-116. Individual areas of the cuticle membrane differ in their relative proportions of 14 proteins, where significant differences between the three quantification criteria (direct, after normalization to ovocledin-17, or to ovocledin-116) were observed in four proteins.

Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization.

The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which are associated with distinct populations of matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The recent development of high-throughput methods has led to the identification of many proteins in the shell, but little is known concerning their role in shell formation. In order to determine precisely the importance of particular proteins relative to eggshell mineralization, this project used qualitative and quantitative proteomics of the uterine fluid constituents, coupled with bioinformatic analysis, to predict the functional role of proteins secreted at each of the three main stages of shell calcification. Besides its relevance to food production and to hen reproduction, eggshell calcification is furthermore a relevant model for studying calcium carbonate biomineralization on a two-dimensional membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressure and temperature.